The present invention relates to the identification and characterization of a promoter of the wheat wcs120 gene. This promoter is inducible by low temperatures in both moncotyledoneous and dicotyledoneous species. It can therefore be used as a universal promoter for genes that are involved in the improvement of low temperature or freeze tolerance in plants.
During the exposure of plants to low temperature (LT), and in the process of cold acclimation (CA) of plants, many physiological and biochemical changes occur, leading in some plants to the development of freezing tolerance (FT). The survival of these tolerant plants at freezing temperatures depends on the timely modulation of specific sets of genes, for which the accumulation of both mRNA and encoded proteins correlate with the development of FT (Guy, C. L. (1990) Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 187-223; Thomashow, M. F. (1990) Adv. Genet. 28: 99-131). A cryoprotective function has been proposed for several low temperature-responsive genes (Houde, M., et al. (1995) Plant J. 8: 583-593; Kurkela, S., et al. (1990) Plant Mol. Biol. 15: 137-144; Sieg, F., et al. (1996) Plant. Physiol. 111:215-221).
The molecular mechanisms governing gene expression at low temperature are not well understood. Recent reports suggest a role for calcium as second messenger in the early events following exposure to chilling temperatures (Knight, H., et al. (1996) Plant Cell 8:489-503; Monroy, A. F., et al. (1995) Plant Cell 7: 321-331). The Ca2+ signal is likely transduced in a series of phosphorylation events which may involve Ca2+-dependent protein kinases. However, there is little information regarding the downstream signalling components leading to the activation of specific sets of genes in response to LT.
Few cis-acting elements responsive to low temperature have been identified so far. In the case of the cor15a gene of Arabidopsis thaliana, Baker, S. S., et al. ((1994) Plant Mol. Biol. 24:701-713) suggested a potential role of G-box-like elements in ABA (abscissic acid) and drought responsiveness. However it is unclear whether these elements also play a role in LT responsiveness. The DR1 core motif (TACCGACAT) in the promoter of the A. thaliana rd29A gene is a cis-element implicated in the response to dehydration, high salt and low temperature (Yamaguchi-Shinozaki. K., et al. (1994) Plant Cell 6:251-264). A similar low-temperature regulatory element (LTRE)(TGGCCGAC), found in the promoter of the Brassica napus BN115 gene, contains the pentamer CCGAC motif which imparts low-temperature responsiveness (White, T. C., et al. (1994b) Plant Physiol. 106:917-928; Jiang, C., et al. (1996) Plant Mol. Biol. 30: 679-684). This motif or similar variants is also present in the promoters of cor15a (Baker, S. S., supra), rd29A (Yamaguchi-Shinozaki, K., supra), Iti78 and Iti65 Arabidopsis genes (Nordin K., et al. (1993) Plant Mol. Biol. 21:641-653) and blt4.6 and blt4.9 barley genes (White A. J, et al. (1994a) J. Exp. Bot. 45:1885-1892).
Several LT-responsive cDNA clones from wheat have been characterized. Among these, the Wcs120 gene is specifically regulated by low temperature (Houde M., et al. (1992) Plant Physiol. 99:1381-1387). The encoded 50 kDa protein is the major member of the WCS120 protein family (Houde, M. (1995), supra). Southern analysis indicates that the gene copy number and gene organization are identical in both freezing tolerant and sensitive wheat cultivars. On the other hand, the accumulation of both Wcs120 mRNA and encoded protein is shown to correlate closely with the differential capacity of wheat cultivars to develop FT (Limin, A. E., et al. (1995) Genome 38:1023-1031). Homologs of Wcs120 and other cold-regulated genes are present in chilling sensitive Gramineae species such as rice and corn, but they are not induced by low temperature (Danyluk, J., et al. (1994) FEBS Lett. 344:20-24). It appears that the expression of the wcs120 gene is regulated mainly at the transcriptional level. It is possible that the inability of some species to CA and to develop FT is due to inefficient cis-acting elements in the promoter or the absence of LT-specific transcription factors.
There thus remains a need to identify the nuclear events regulating the cold-specific expression of the Wcs120 gene. It therefore appeared of great interest to characterize the promoter region of the wheat wcs120 gene and to analyse the role of the promoter in the development of FT in plants.
The present invention seeks to meet these and other needs.
The invention concerns the identification of nuclear events regulating cold-specific gene expression involved in CA of plants in the development of FT. Specifically, it is the characterization of a gene promoter in plants that is induced in response to low temperature to express genes involved in the CA of plants. The promoter of this invention was first isolated from the 5xe2x80x2 start site of the wheat wcs120 gene, sequenced and analysed for specific elements or motifs. A comparison of homologous function in promoters in other genes that are expressed in response to adverse environmental conditions, in the same and different species has shown conservation of some of these motifs. Further, by deletion analyses of the promoter, the functions of specific promoter regions are characterized and their importance in the response to the development of FT is identified. The invention additionally teaches the ability of the promoter to be used as a universal promoter in driving genes in response to low temperature involved in CA in dicots and monocot plant species for improvement of FT.
The invention additionally relates to the nuclear factors and events involved in the expression of the wheat low-temperature responsive gene wcs120. The results demonstrate that these nuclear factors regulate the expression of wcs120 at the level of the promoter by differential binding of these factors to the promoter under different environmental conditions.
The inventors are the first to isolate, identify, sequence and characterize the promoter of the wheat wcs120 gene. The inventors are also the first to characterize the promoter and functional variants, fragments and derivatives thereof by deletion analyses.
The inventors are the first to demonstrate that this promoter can be induced by low-temperatures in all plants, those that are cold-sensitive and cold-tolerant, and, in monocot and dicots plant species, thus demonstrating the universality of the promoter in inducing homologous and heterologous gene expression in response to low temperature.
The inventors are also the first to demonstrate the role of nuclear factors in regulating the transcription of the wheat wcs120 gene in response to different temperatures.
Before the present invention, it was believed that sensitivity to low temperature and inability to CA in plants was due to genetic variability in the low temperature responsive genes. The inventors are the first to demonstrate that the level of repression of the cold-induced genes, specifically wcs 120, is not due to variations in the gene itself or its promoter but rather to nuclear factors acting at the level of the promoter.
The promoter of the present invention and any derivatives or fragments thereof, can therefore be used in the design of transgenic plants in need of improving resistance to low temperatures. This can be accomplished by replacing the homologous promoter with the promoter of the present invention or any derivatives or fragments thereof, to drive the expression of the genes needed to cold-acclimate a plant and in this manner improve the FT of the plant.
In accordance with the present invention, there is therefore provided, a sequence of the promoter region of the wheat wcs120 gene.
In accordance with another aspect of the present invention, there is also provided the promoter, derivatives or fragments thereof being universal, such that the promoter, derivatives or fragments thereof are capable of driving the expression of genes in response to low temperature in plants including, monocot and dicots, and, cold-sensitive and cold-tolerant species.
In accordance with another aspect of the present invention, there is provided, a use for the promoter of the instant invention in transgenic plants, monocot and dicots plant species, such that the construct comprises the promoter, derivatives or fragments thereof to drive the expression of genes in response to low temperatures in different plant species.
In accordance with yet another aspect of the present invention, there are provided elements within the promoter that have homologies with other promoter elements from other genes. There is also provided regions of the promoter that are the minimal size fragments, derivatives or variants thereof capable of responsiveness to low temperature.
The sequences and polypeptides useful to practice the invention include without being limited thereto, mutants, homologs, subtypes, alleles and the like. It shall be understood that generally, the sequences of the present invention should encode a functional catalytic and interaction domain. It will be clear to the person of ordinary skill that whether an interaction or catalytic domain of the present invention, variant, derivative, or fragment thereof retains its function can be readily determined by using the teachings and assays of the present invention and the general teachings of the art.
As used herein, the designation xe2x80x9cvariantxe2x80x9d denotes, in the context of a variant of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. This variant or equivalent may be from the same or different species and may be a natural variant or be prepared synthetically. Such variants include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to variants of nucleic acid sequences which can have substitutions, deletions or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid as chemico-physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like.
The term xe2x80x9cderivativexe2x80x9d is intended to include any of the above described variants that have been used for the purpose of labelling, binding or are comprised in fusion product(s).
The term xe2x80x9cfragmentxe2x80x9d refers to any segment of an identified DNA, RNA or amino acid sequence and/or any segment of any of the variants or derivatives described in the above definitions.
Thus, the term xe2x80x9cvariantxe2x80x9d refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.
The terms xe2x80x9cvariantsxe2x80x9d, xe2x80x9cderivativesxe2x80x9d and xe2x80x9cfragmentsxe2x80x9d of the present invention refer herein to proteins or nucleic acid molecules which can be isolated/purified, synthesized chemically or produced through recombinant DNA technology. All these methods are well known in the art.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted as limiting the scope of the present invention.